1. Field of the Invention
The present invention is directed to the field of immunotherapy and more particularly to a glycoconjugate, a composition and vaccine comprising the same and to the use thereof for enhancing the immune response and notably in cancer therapy and in therapeutic of infection caused by pathogenic agent against whom a humoral or a cellular immune response is necessary. The invention relates also to a diagnosis kit and a method for diagnosis of cancer.
2. Prior Art/Relevant Literature
As a result of aberrant glycosylation, cancer-associated carbohydrate antigens are exposed at the surface of tumor cells whereas they are hidden in normal cells (Bhavananda, et al. (1991)). Recent advances in immunology and in the identification of tumor specific antigens have renewed the interest for the development of cancer vaccines, and these exposed glycosidic B-cell epitopes have been considered as attractive targets for immunotherapy named xe2x80x9cActive Specific Immunotherapyxe2x80x9d (ASI) by Longenecker (MacLean et al; (1994)). This approach involves immunization with a defined antigen to elicit a specific immune response to that antigen and could represent an alternative to the conventional cancer therapies.
Among the large number of known tumor markers, the Tn (xcex1-GalNAc-Ser/Thr), the T* (xcex2-Cal-(1xe2x86x923)-xcex1-GalNAc Ser/Thr) and the sialosyl-Tn (xcex1-NeuAc-(2xe2x86x926)-xcex1-GalNAc-Ser/Thr) antigens have been extensively studied since they are expressed on mucin-type glycoproteins by the majority of adenocarcinomas (Springer et al. (1984). Indeed, several studies have shown some protection against tumors after immunization with these glycosidic antigens, in experimental or clinical studies. These tumour associated carbohydrates are relevant markers for cancer diagnostic and prognosis (Itzkowitz et al. (1990)). Using desialylated red blood cells, which are rich in T and Tn determinants, Springer observed a long-term effective protection against recurrence of human breast carcinoma (Springer et al. (1995), Springer et al. (1994). An other group investigated the potential of ASI with desialylated ovine submaxillary mucin (d-OSM), which contains high density of the Tn epitope; their studies showed that this antigen provided a good protection and a long-term survival in mice with mammary carcinoma (Singhal et al. (1991)). Partially d-OSM also gave efficient protection against human colon carcinoma (O""Boyle et al. (1992)). Ratcliffe et al. were the first to use a synthetic tumor-associated antigen, a T antigen-protein conjugate, to stimulate an efficient immune response in rabbits (Ratcliff et al. (1981)). Thereafter Longenecker extensively studied similar synthetic carbohydrate hapten conjugates and found that they induce an increased survival of mice grafted with mammary carcinoma cells (Fung et al. (1990)), and of patients with ovarian cancers (MacLean et al. (1992)). Similar studies of the same group have further shown an increased protection of patients suffering from breast cancer (Longenecker et al. 1993)) or melanoma (Helling et al. (1995)) after respective administration of sialosyl Tn or the GM2 ganglioside-protein conjugates. On the other hand, Toyokuni et al. generated an anti-tumor antibody response in mice after immunization with a Tn antigen coupled either to OSA (Ovine Serum Albumin) or to a synthetic lipopeptide (Toyokuni et al. (1994)). This last result was interesting since it was the first example of a small synthetic carbohydrate antigen that generates an Immune response against a tumor associated carbohydrate antigen, without the use of a macromolecular carrier or adjuvants.
These studies suggested that carbohydrate antigens are appropriate candidates for anti-tumor vaccine development. However, carbohydrate antigens do not possess T-cell epitope and therefore induce only weak T cell-independent antibody response. Several approaches have been explored to increase the immunogenicity of such carbohydrates. The use of biological material which expresses clusters of antigens on a protein backbone (like desialylated red blood cells or OSM) is a possibility. But the most widely used approach is to conjugate the carbohydrate to a carrier protein, such as Bovine serum albumin (BSA) or Keyhole limpet hemocyanin (KLH).
Although these immunogens have shown some promise, protein carriers display major disadvantages. The grafted epitope represents only a small part of the total conjugate and it is distributed at random on the carrier surface. Therefore, immune responses to the carrier molecule may result in a low level of the desired antibodies as compared to the total amount of antibodies produced. Moreover, these conjugates present ambiguity in both composition and structure and they do not always induce reproducible immune response. Recent advances in the total synthesis of oligosaccharides expressed by tumour cells (Deshpande et al. (1997), Sames et al. (1997)) open new possibilities for such achievement. However, haptenic molecules such as carbohydrates require their association in more complex structures to stimulate immune responses. The use of traditional protein conjugates raises the problem of hapten-specific suppression (Herzenberg et al. (1980), Schutze et al. (1985)), and their poorly defined chemical composition and structure may limit their efficacy.
Until now, as for chemically refined structures dendrimeric poly-lysine backbones, which will be described in more detail later in the present specification, have been widely used for presenting peptides (Tam et al., 1994). However, to our knowledge, there is only one preliminary attempt of their utilisation for presenting carbohydrates to the immune system (Roy et al., 1994). This latter reference teaches the synthesis of three sialylated multiple antigen peptides having tetanus toxin T-cell epitopes. A similar strategy was also recently published (Lett, et al. (1995), CHONG et al.,1997) where the authors couple mixtures of natural or synthetic polysaccharides to a Multiple Antigenic Peptidic system.
Thus, there still exist a need for a new conjugate circumventing the drawbacks mentioned above of the prior art constructions which has a chemically defined structure, is capable of stimulating both the antibody response and the T response when administered in a human or animal body, while avoiding undesired immune responses.
Accordingly, the present invention is generally directed to a carbohydrate peptide conjugate comprising:
a carrier comprising a dendrimeric poly-Lysine enabling multiple epitopes to be covalently attached thereto,
at least one peptide comprising one T epitope or several identical or different T epitopes
at least one carbohydrate moiety containing B epitope, provided it is not a sialoside, or several identical or different B epitopes.
The peptide comprising the T epitope(s) can be bound to a lysine of said carrier, as the carbohydrate moiety containing B epitope(s).
This approach for presenting epitopes is herein referred to as the Multiple Antigen Glycopeptide (MAG). The conjugate of the present invention is notably useful for enhancing the antibody response in a human or animal body to which it has been administered and in particular as a vaccine.
Moreover, since a multiple antigenic O-linked glycopeptide (MAG), according to the present invention, carrying for example the carbohydrate Tn antigen associated with a CD4+ T cell epitope was shown able to induce anti-Tn IgG antibodies which recognize human tumour cell lines, accordingly the present invention also concerns an composition capable of increasing the survival of a tumour-bearing human or animal. A therapeutic immunisation protocol performed with this fully synthetic immunogen increased the survival of tumour bearing mice.
More particularly the present invention is directed to a carbohydrate peptide conjugate comprising:
at least 3 lysine residues covalently bound to each other,
at least one peptide comprising a T epitope bound to a lysine residue, and
at least one carbohydrate moiety containing epitope B, optionally substituted, covalently linked to the end of said peptide opposite to lysine, and with the proviso that said carbohydrate moiety is not a sialoside radical.
According to another embodiment of the invention, the conjugate comprises:
at least one peptide comprising one T epitope, or several identical or different T epitopes, and
at least one carbohydrate moiety, or a derivative thereof, containing B epitope, provided it is not sialoside, or several identical of different epitopes.
In a further embodiment of the invention, the carbohydrate peptide conjugate is linear.
Another object of the present invention is a pharmaceutical composition comprising the conjugate of the present invention.
A further object of the present invention is a vaccine comprising the conjugate according to the present invention.
A still further object of the present invention is a method of enhancing the immune response of a human or animal body, in particular B and/or T-cell responses, wherein the conjugate according to the present invention is administered to said human or animal body.
Another object of the present invention is a method of inducing B-cell responses against saccharidic epitopes in a human or animal body, wherein the conjugate according to the invention is administered to said human or animal body.
A still further object of the present invention is a method of vaccination of a human or animal body wherein the conjugate according to the present invention is administered to said human or animal body.
Another object of the present invention is a diagnosis kit comprising antigen specific antibodies elicited by immunization of a human or animal body with a composition according to the present invention.
A further object of the present invention is a method of diagnosis of cancer wherein a biological sample is brought into contact with at least one of these antibodies and wherein one determines the formation of complexes between this antibody and molecules comprised in the said sample.
A still further object is an immunogenic composition as described hereabove, capable to elicit an immune response against a viral infection caused by a pathogen such as hepatitis virus, HIV or CMV.
The present invention will now be described in details in the following description with reference to the drawings below.